Recently there have been important discoveries of potent and broadly neutralizing monoclonal antibodies (bnMAb) isolated from chronically HIV-infected individuals. It will likely follow that attempts will be made to re-elicit such antibody responses through the rational design of immunogens based on these discoveries. Despite having potent and broad neutralizing activity, the serum used to generate these bnMAbs did not however produce sterilizing immunity in the infected individuals from which they were isolated nor did they neutralize all of the Tier 1-2 viruses that were tested, suggesting potential escape variants. Therefore, the goal of this proposal is to characterize escape from these broad and potent neutralizing antibodies. The major aims are to (1) map the escape pathways used by a panel of primary acute and chronic HIV molecular clones in the development of resistance to these bnMAbs, (2) determine whether escape from one broadly neutralizing antibody changes the sensitivity to a different broadly neutralizing antibody due to global changes in the HIV envelope glycoprotein (Env) structure, and (3) understand whether the escape mutation(s) alter(s) the fitness of the resulting variant. To accomplish these aims, a panel of HIV clones that express early and late time-point Envs will be serially-passaged in the presence of bnMAbs (i.e. VRC01, VRC03, PG9 and PG16) to drive immunological escape. Then, sequential samples will be analyzed at each passage of the virus to chart the (i) accumulation of genetic mutations and (ii) development of resistance. Once escape mutants are generated and identified, neutralization assays with each escape mutant using the other bnMAbs will be performed. Moreover, escape mutant Envs will be subcloned into a replication-competent HIV background to determine whether the mutation(s) also affect the fitness of the virus. The information gained in this study is highly relevant to rational immunogen design, since it (i) deepens the understanding of the mechanisms used by primary Envs to escape from bnMAbs, (ii) may identify second-site and/or off-target mutations in Env that are involved in escape from bnMAbs, and (iii) may provide an estimate of the number of epitope targets that are needed to afford protective immunity against not only the transmitted founder virus, but also its possible escape variants. In the aggregate, this project is highly relevant to an understanding of the requirements for an effective vaccine that could prevent acquisition of HIV/AIDS.